Cell surface expression vector of sars virus antigen and microorganisms transformed thereby

ABSTRACT

The present invention relates to a surface expression vector of SARS coronavirus antigen containing a gene encoding an antigen of SARS inducing coronavirus and any one or two or more of genes pgsB, pgsC and pgsA encoding poly-gamma-glutamic acid synthase complex, a microorganism transformed by the surface expression vector, and a SARS vaccine comprising the microorganism. According to the present invention, it is possible to economically produce a vaccine for prevention and treatment of SARS using a recombinant strain expressing an SARS coronavirus antigen on their surface.

TECHNICAL FIELD

The present invention relates to a vector expressing antigens of SARS onthe surface of a microorganism, a microorganism transformed by thevector, and a vaccine for prevention of SARS comprising the transformedmicroorganism or an extracted and purified substance thereof. Moreparticularly, it relates to a surface expression vector containing agene encoding antigen proteins of SARS inducing coronavirus and any oneor two or more of genes pgsB, pgsC and pgsA encoding poly-gamma-glutamicacid synthase complex which is a microorganism surface anchoring motif,a microorganism transformed by the vector, and a SARS vaccine comprisingthe transformed microorganism as an effective ingredient.

BACKGROUND ART

Severe Acute Respiratory Syndrome (SARS) is a new type of an epidemicwhich has spread all over the world including Hong Kong, Singapore,Canada (Toronto) and so forth since it firstly broke out in November2002 centering around Guangdong province in China. It shows respiratorysymptoms such as fever of 38° C. or higher and coughing, dyspnoea,atypical pneumonia. The agent of SARS is known as a mutant pathogeniccoronavirus.

Generally, the members of coronavirus family are very large RNA viruseshaving (+)RNA. The genome is composed of about 29,000 to 31,000 basesand observed as a crown shape under a microscope. It contributes toupper respiratory diseases in human, respiratory, liver, nerves andintestines related diseases in animals. Three groups of coronavirusexist in nature. Among them, group I and group II infect mammals andgroup III infects birds.

The known coronavirus in nature sometimes induce lung related diseasesin persons with weakened immune system or cause severe diseases inanimals such as dogs, cats, pigs, mice, birds and the like. They show avery high mutation rate and a high recombination rate of about 25%. Itis presumed that such properties cause mutation of original coronavirus,to produce a novel mutant coronavirus (SARS coronavirus), which ispropagated from animals to human.

According to World Health Organization (WHO), 7,447 suspected SARSpatients in 31 countries have identified since November, 2002 and 551 ofthem died. The SARS infection danger zone of 2003 include Beijing,Guangdong, Hong Kong, inner Mongolia, Shanxi and Tianjin in China,Singapore, Toronto in Canada, Taiwan, Ulanbaator in Mongol, Philippinesand the like. However, this has a risk to be spread all over the world.

Since the outbreak on 2002, as to SARS coronavirus, a Germany institutefor tropical medicine firstly performed decoding of the nucleotidesequence of SARS virus. The research team decoded the nucleotidesequence of a specific genetic part where the amplification by PCR(Polymerase Chain Reaction) can be done. The decoded result was given toArtus GmbH which is a bioengineering company in Germany and used todevelop a kit to detect infection of SARS. This kit can determine theinfection of SARS virus by amplification of virus gene from a suspectedSARS patient.

Thereafter, the whole genome of SARS virus was decoded and up to now,the sequences of more than 12 isolate strains are completely analyzed.The whole sequence of Urbani strain, which is the firstly isolatedstrain [dubbing the name of the WHO mission doctor who died of SARS,SARS-Cov strain (Rota, Pa., Science 108:5952, 2003; GenBank AccessionAY278741)] was decoded by a CDC research team of USA. The Canada BritishColumbia Cancer search center team analyzed the whole sequence of SARSTor2 virus strain isolated from a patient in Toronto, Canada, on Apr.12, 2003 (Marra, M. A., Science 108:5953, 2003; GenBank Accession274119).

Though the two research teams analyzed coronavirus isolated frompatients infected with SARS in each different place, the two virusesshowed difference in only 15 bases. This suggests that SARS has beeninduced from the same virus. Also, according to the result of a genomicanalysis of SARS coronavirus, it is known that it has the samecomponents forming proteins as those of the existing coronavirus butshows little homology in genome and amino acids by genome. Rat hepatitisvirus and turkey bronchitis virus show similarity to SARS coronavirus.However, the correlation of SARS coronavirus and other coronavirus ispresented by molecular taxonomic analysis and it is concluded that SARScoronavirus is different from the existing groups.

At present, the detection of SARS coronavirus begins with PCR and thepositive result of the antibody test is determined by ELISA or IFA. Thevirus isolation is performed by subjecting a subject identified by PCRto a cell culture test and determining the infection of SARScoronavirus.

There is no fundamental method for treating SARS but supplementarysupporting therapy. The research on SARS coronavirus, which is an agentof the new epidemic, is in the beginning step and no vaccine forprevention was developed. Diversified researches are being conducted todevelop a vaccine for prevention all over the world.

The technology to attach and express a desired protein onto the cellsurface of a microorganism is called as cell surface display technology.The cell surface display technology uses surface proteins ofmicroorganisms such as bacteria or yeast as a surface anchoring motif toexpress a foreign protein on the surface and has an application scopeincluding production of recombinant live vaccine, construction ofpeptide/antibody library and screening, whole cell absorbent, whole cellbiotransformation catalyst and the like. The application scope of thistechnology is determined by a protein to be expressed on the cellsurface. Therefore, the cell surface display technology has tremendouspotential of industrial applicability.

For successive cell surface display technology, the surface anchoringmotif is the most important. It is the core of this technology to selectand develop a motif expressing a foreign protein on the cell surfaceeffectively.

Therefore, in order to select a surface anchoring motif, the followingproperties should be considered. (1) It should have a secretion signalto help a foreign protein to pass through the cellular inner membrane sothat the foreign protein can be transferred to the cell surface. (2) Itshould have a target signal to help a foreign protein to be stably fixedon the surface of the cellular outer membrane. (3) It can be expressedin a large quantity on the cell surface but does not affect growth ofthe cell. (4) It has nothing to do with protein size and can express aforeign protein without change in the three-dimensional structure of theprotein. However, a surface anchoring motif satisfying the foregoingrequirements has not yet been developed.

The surface anchoring motives which have been known and used so far arelargely classified into four types of cell outer membrane proteins,lipoproteins, secretory proteins, surface organ proteins such asflagella protein. In case of gram negative bacteria, proteins existingon the cellular outer membrane such as LamB, PhoE (Charbit et al., J.Immunol., 139:1658, 1987; Agterberg et al., Vaccine, 8:85, 1990), OmpAand the like have been used. Also, lipoproteins such as TraT (Felici etal., J. Mol. Biol., 222:301, 1991), PAL (peptidoglycan associatedlipoprotein) (Fuchs et al., Bio/Technology, 9:1369, 1991) andLpp(Francisco et al., Proc. Natl. Acad. Sci. USA, 489:2713, 1992) havebeen used. Fimbriae proteins such as FimA or FimH adhesion of tppe 1fimbriae (Hedegaard et al., Gene, 85:115, 1989), pili proteins such asPapA pilu subunit have been used as a surface anchoring motif to attemptexpression of a foreign protein. In addition, it has been reported thatice nucleation protein (Jung et al., Nat. Biotechnol., 16:576, 1998;Jung et al., Enzyme Microb. Technol., 22:348, 1998; Lee et al., Nat.Biotechnol., 18:645, 2000), pullulanase of Klebsiela oxytoca (Kornackeret al., Mol. Microl., 4:1101, 1990), IgA protease of Neiseria (Klauseret al., EMBO J., 9:1991, 1990), AIDA-1, which is adhesion of E. coli,VirG protein of shigella, a fusion protein of Lpp and OmpA may be usedas a surface anchoring motif. Upon use of gram positive bacteria, therehave been reported that malaria antigen was effectively expressed usingStaphylococcus aureus derived protein A and FnBPB protein as a surfaceanchoring motif, a surface coat protein of lactic acid bacteria used insurface expression, and surface proteins of gram positive bacteria suchas Streptococcus pyogenes derived M6 protein (Medaglini, D et al., Proc.Natl. Acad. Sci. USA., 92:6868, 1995), Bacillus anthracis derivedS-layer protein EA1, Bacillus subtilis CotB and the like were used as amotif.

The present inventors have developed a novel vector for effectivelyexpressing a foreign protein on the cell surface of a microorganism byusing poly-gamma glutamic acid synthesizing complex gene (pgsBCA)derived from Bacillus genus strain as a novel surface anchoring motifand a method for mass-expressing a foreign protein on the surface of amicroorganism transformed by the vector (Korean Patent Application No.10-2001-48373).

Researches have been conducted to stably express a pathogenic antigen oran antigen determining group in bacteria suitable for mass-production bygenetic engineering method using the above-listed surface anchoringmotives. Particularly, it has been reported that an exogenous immunogenexpressed on the surface non-pathogenic bacteria, when being orallyadministered in the live state, can induce more sustained and strongerimmune response, as compared to vaccines using attenuated pathogenicbacteria or viruses. Such induction of immune response is attributableto the adjuvant action of the surface structures of bacteria to increaseantigenicity of the foreign protein expressed on the surface and immuneresponse to the live bacteria in the living body. The development of arecombinant live vaccine of non-pathogenic bacteria using this surfaceexpression system has attracted public attention.

Therefore, the present inventors have succeeded in mass-expressingantigens of SARS coronavirus chosen by gene and protein analyses on thesurface of a non-pathogenic microorganism, of which food safety issecured, such as lactic acid bacteria by using poly-gamma-glutamic acidsynthesizing complex gene (pgsBCA) derived from Bacillus genus strain asa surface anchoring motif and developed an economic and stable vaccineto induce production of antibody to SARS coronavirus in blood andmucosal immunization through oral administration of the microorganism.

DISCLOSURE OF INVENTION

Therefore, it is an object of the present invention to provide a vectorcapable of expressing a SARS coronavirus antigen by employing a surfaceexpression system of a microorganism and a microorganism transformed bythe vector.

It is another object of the present invention to provide a transformedmicroorganism having an antigen of SARS coronavirus expressed on thesurface, a vaccine for prevention of SARS comprising a SARS coronavirusantigen extracted from the microorganism or a SARS coronavirus antigenpurified from the microorganism as an effective ingredient.

In order to accomplish the above objects, according to the presentinvention, there is provided a surface expression vector comprising anyone or two or more of pgsB, pgsC and pgsA genes encodingpoly-gamma-glutamic acid synthase complex and a gene encoding a spikeantigen protein or a nucleocapsid antigen protein of SARS coronavirus.

According to the present invention, as the surface antigen protein gene,any gene encoding a spike antigen protein of SARS coronavirus can beused. It is possible to use a spike antigen protein gene of SARScoronavirus alone or as a complex of two or more. Also, the geneencoding the poly-gamma-glutamic acid synthase complex preferablyincludes pgsA. The spike antigen protein may be SARS SA, SARS SB, SARSSC, SARS SD or SARS SBC and the nucleocapsid antigen protein may be SARSNA, SARS NB or SARS N.

Also, the present invention provides a microorganism transformed by theexpression vector and a method for producing a spike antigen protein ora nucleocapsid antigen protein of SARS coronavirus comprising culturingthe microorganism.

The microorganism applicable to the present invention may be anymicroorganism which does not show toxicity upon application to a livingbody, or any attenuated microorganism. For example, it can be properlyselected from gram negative bacteria, such as E. coli, Salmonella typhi,Salmonella typhimurium, Vibrio cholerae, Mycobacterium bovis, Shigellaand the like or gram positive bacteria such as Bacillus, Lactobacillus,Lactococcus, Staphylococcus, Listeria monocytogenes, Streptococcus andthe like. Selection of an edible microorganism such as lactic acidbacteria is particularly preferred.

Further, the present invention provides a vaccine for prevention of SARScomprising a microorganism having the antigen protein expressed on thesurface, a crude form extracted from cell membrane components of themicroorganism which has been broken, or an antigen protein purified fromthe microorganism as an effective ingredient.

The vaccine according to the present invention can be used as a medicinefor prevention of SARS (Severe Acute Respiratory Syndrome) induced bySARS coronavirus.

The vaccine according to the present invention can be taken by oraladministration or in food, subcutaneously or intra-peritoneallyinjected, or administered by the intranasal route.

Up to date; the infection of SARS coronavirus is known to be induced byinfection of a respiratory organ by infectious droplets and presumed tooccur at the mucosal surface of the respiratory organ. Thus, theprotection of infection by mucosal immunity is very important. Since themicroorganism expressing an antigen of SARS coronavirus on the surfacehas an advantage that can more effectively induce antibody formation ona mucous membrane (mucosal response), the vaccine for oraladministration or the vaccine for intranasal administration using thetransformed microorganism is expected to be more effective than aparenteral vaccine in the protection against SARS coronavirus.

BRIEF DESCRIPTION OF DRAWINGS

Further objects and advantages of the invention can be more fullyunderstood from the following detailed description taken in conjunctionwith the accompanying drawings.

FIG. 1 shows the relations between four antigenic sites (A, B, C, D) ofswine transmissible gastro enteritis virus and the spike protein of SARScoronavirus by hydrophilicity plot according to the Kyte-Doolittlemethod, antigenic index according to the Jameson-wolf method and surfaceprobability plot according to the Emini method.

FIG. 2 shows the relation between the nucleocapsid protein of swinetransmissible gastro enteritis virus and the nucleocapsid protein ofSARS coronavirus by hydrophilicity plot according to the Kyte-Doolittlemethod, antigenic index according to the Jameson-wolf method and surfaceprobability plot according to the Emini method.

FIG. 3A is a genetic map of the vector pHCE2LB:pgsA-SARS SA for surfaceexpression comprising the gram negative and gram positive microorganismsas a host according to the present invention, FIG. 3B is a genetic mapof pHCE2LB:pgsA-SARS SC according to the present invention and FIG. 3Cis a genetic map of pHCE2LB:pgsA-SARS SBC according to the presentinvention.

FIG. 4A is a genetic map of the vector pHCE2LB:pgsA-SARS NB according tothe present invention and FIG. 4B is a genetic map of pHCE2LB:pgsA-SARSN according to the present invention.

FIGS. 5A, 5B and SC are to identify expression of the SARS SA, SARS SCand SARS SBC antigens fused with the cellular outer membrane proteinpgsA in Lactobacillus by showing the specific bonding between a specificantibody to pgsA and the fusion proteins by Western immunoblotting.

FIGS. 6A and 6B are to identify surface expression of the SARS SA andSARS SBC antigens fused with the cellular outer membrane protein pgsA inLactobacillus by performing Western immunoblotting using proteinsfragmented from lactic acid bacteria cells as a specific antibody topgsA and FIG. 6C is to identify surface expression of the SARS SBCantigen in Lactobacillus by FACScan assay.

FIGS. 7A and 7B are to identify surface expression of the SARS NB andSARS N antigens fused with the cellular outer membrane protein pgsA inLactobacillus by performing Western immunoblotting using proteinsfragmented from lactic acid bacteria cells as a specific antibody topgsA.

FIG. 8 shows the results of measurement of IgG antibody value to theSARS SA and SARS SC antigens in serum of mouse which has been orally andintranasally administered with the Lactobacillus casei strains, whichare each transformed with the vectors pHCE2LB:pgsA-SARS SA,pHCE2LB:pgsA-SARS SC and pHCE1LB:pgsA-SARS NB for surface expressionaccording to the present invention and have the surface expression ofthe antigen group identified by ELISA (Enzyme-linked ImmunosorbentAssay).

FIG. 9 shows the results of measurement of IgA antibody value to theSARS SA and SARS SC antigens in the intestine washing liquid andbronchus-alveolar washing liquid of mouse which has been orally andintranasally administered with the Lactobacillus casei strains, whichare each transformed with the vectors pHCE2LB:pgsA-SARS SA,pHCE2LB:pgsA-SARS SC and pHCE1LB:pgsA-SARS NB for surface expressionaccording to the present invention and have the surface expression ofthe antigen group identified, by ELISA.

FIG. 10 shows the results of measurement of IgG antibody value to theSARS NB antigen group in serum of mouse which has been orally andintranasally administered with the Lactobacillus casei strains, whichare each transformed with the vectors pHCE2LB:pgsA-SARS SA,pHCE2LB:pgsA-SARS SC and pHCE1LB:pgsA-SARS NB for surface expressionaccording to the present invention and have the surface expression ofthe antigen group identified, by ELISA.

FIG. 11 shows the results of measurement of IgA antibody value to theSARS NB antigen group in the intestine washing liquid andbronchus-alveolar washing liquid of mouse which has been orally andintranasally administered with the Lactobacillus casei strains, whichare each transformed with the vectors pHCE2LB:pgsA-SARS SA,pHCE2LB:pgsA-SARS SC and pHCE1LB:pgsA-SARS NB for surface expressionaccording to the present invention and have the surface expression ofthe antigen group identified, by ELISA.

BEST MODE FOR CARRYING OUT THE INVENTION

The Now, the present invention will be explained in further detail bythe following examples. It is apparent to those possessing ordinaryknowledge in the art that the examples are only for concrete explanationof the present invention and the scope of the present invention is notlimited thereto.

Particularly, though genes of an antigenic site in the spike protein ofSARS coronavirus and genes of an antigenic site in the nucleocapsidprotein of SARS coronavirus are applied in the following examples, anyantigen protein gene may be used alone or as a complex of two or more.

Also, in the following examples, the gene pgsBCA of the cellular outermembrane protein which is involved in synthesis of poly-gamma-glutamicacid is obtained from Bacillus subtilis var. chungkookjang (KCTC 0697BP)and used. However, according to the present invention, the gene includesvectors prepared using pgsBCA obtained from all Bacillus genus strainsproducing poly-gamma-glutamic acid or microorganisms transformed withthose vectors. For example, preparation of a vector for a vaccine usingthe pgsBCA gene derived from other strains having homology of 80% ormore with the sequence of the pgsBCA gene existing in Bacillus subtilisvar. chungkookjang and use of the vector are included in the scope ofthe present invention.

Further, in the following examples, only pgsA of the gene pgsBCA is usedto construct a vector for surface expression. However, as can beinferred from indirect examples, use of the whole or a part of the genepgsBCA to construct a vector for a vaccine is included in the scope ofthe present invention.

In the following examples, Salmonella typhi, which is a gram negativebacterium and Lactobacillus, which is a gram positive bacterium are usedas a host for the vector. However, it becomes apparent to those skilledin the art that any kind of gram negative bacteria or gram positivebacteria which have been transformed by the method according to thepresent invention can provide the same results.

In addition, in the following examples, only cases applying amicroorganism itself transformed by the vector for a vaccine accordingto the present invention as a live vaccine to a living body arepresented. However, according to the knowledge of the vaccine-relatedtechnical field, it is natural to have identical or similar results evenwhen expression proteins (antigen proteins of SARS coronavirus) crudelyextracted from the microorganism or purified expression proteins areapplied to a living body.

EXAMPLE 1 Synthesis of Antigenic Site Gene in Spike Protein of SARSCoronavirus

The spike protein of SARS coronavirus is a glycoprotein composed of 1256amino acids. In case of other coronavirus which have been much examined,the spike protein is mostly inserted into an envelope protein. coveringthe surface of a virus particle to have a structure exposed to theoutside. The exposed site and the antigenic site have been intensivelystudied as a target antigen of a vaccine to induce virus infection andto prevent the infection.

Therefore, in order to select a site capable of showing antigenicityfrom the 1256 amino acids of the spike protein of SARS coronavirus, theantigenic site was chosen by comparative analysis of proteins andstructural comparative analysis with the spike protein of other swinetransmissible gastroenteritis (TGE) coronavirus which has been studiedfor antigenicity and synthesized. Concretely, the antigenic site of thespike protein of swine transmissible gastroenteritis virus is well knownas four sites (A, B, C, D) (Enjuanes, L., Virology, 183:225, 1991). Therelation between these sites and the spike protein of SARS coronaviruswas analyzed by hydrophilicity plot according to the Kyte-Doolittlemethod, antigenic index according to the Jameson-wolf method and surfaceprobability plot according to the Emini method and SARS SA, SARS SB,SARS SC and SARS SD were selected from the sequence of the spike proteinof SARS coronavirus Tor2 isolate (FIG. 1).

Firstly, based on the sequence of the spike protein of SARS coronavirusTor2 isolate (21492-25259 bases, 1255 amino acids), of which the wholesequence had been identified, the 2 to 114 amino acid site which wasexpected to be an antigenic site was selected and denominated SARS SA,the 375 to 470 amino acid site was selected and denominated SARS SB, the510 to 596 amino acid site was selected and denominated SARS SC, and the1117 to 1197 amino acid site was selected and denominated SARS SD. Amongthese antigenic sites, genes of the SARS SA and SARS SC sites weresynthesized.

In order to synthesize a gene corresponding to the 113 length aminoacids denominated SARS SA, PCR was performed using primers of SEQ IDNOs: 1 to 8 to obtain the amplified SARS SA gene of 339 bp. SEQ ID NO:1: 5′-ggatcctttattttcttattatttcttactctcactagtggtagtgaccttgaccg-3′ SEQ IDNO: 2: 5′-tgagtgtaattaggagcttgaacatcatcaaaagtggtacaacggtcaaggtc-3′ SEQID NO: 3:5′-aattacactcaacatacttcatctatgcgtggggtttactatcctgatgaaatttttc-3′ SEQ IDNO: 4: 5′-aaaatggaagaaataaatcctgagttaaataaagagtgtctgaacgaaaaattt-3′ SEQID NO: 5:5′-cttccattttattctaatgttactgggtttcatactattaatcatacgtttggcaac-3′ SEQ IDNO: 6: 5′-ggcagcaaaataaataccatccttaaaaggaatgacagggttgccaaacgtatg-5′ SEQID NO: 7: 5′-atttattttgctgccacagagaaatcaaatgttgtccgtggttgggtttttgg-3′SEQ ID NO: 8:5′-ggtaccaagcttattacacagactgtgacttgttgttcatggtagaaccaaaaaccc-3′

In order to synthesize a gene corresponding to the 87 length amino acidsdenominated SARS SC, PCR was performed using primers of SEQ ID NOs: 9 to14 to obtain the amplified SARS SC gene of 261 bp. SEQ ID NO: 9:5′-ggatccgtttgtggtccaaaattatctactgaccttattaagaaccagtgtgtcaat-3′ SEQ IDNO: 10: 5′-gaagaaggagttaacacaccagtaccagtgagaccattaaaattaaaattgacacact-3′SEQ ID NO: 11:5′-aactccttcttcaaagcgttttcaaccatttcaacaatttggccgtgatgtttctga-3′ SEQ IDNO: 12: 5′-ctaaaatttcagatgttttaggatcacgaacagaatcagtgaaatcagaaacat-3′ SEQID NO: 13: 5′-ctgaaattttagacatttcaccttgtgcttttgggggtgtaagtgtaattaca-3′SEQ ID NO: 14:5′-ggtaccaagcttattaaacagcaacttcagatgaagcatttgtaccaggtgtaattac-3′

In addition, the genes of the antigenic sites were obtained bysynthesis, a gene encoding the site of 264 to 596 amino acids wasamplified by PCR using the SARS spike cDNA clone (SARS coronavirus TOR2)from Canada's Michael Smith Genome Science Center as a template andprimers of SEQ ID NOs: 15 and 16 to obtain a gene of 996 bp, which wasdenominated SARS SBC [this gene contains a critical site to produce aneutralizing antiby (PNAS, 101:2536, 2004)]. SEQ ID NO: 15 (SBC sense):5′-cgcggatccctcaagtatgatgaaaat-3′ SEQ ID NO: 16 (SBC anti-sense):5′-cggggtaccttaaacagcaacttcaga-3′

EXAMPLE 2 Synthesis of Antigenic Site Gene in Nucleocapsid Protein ofSARS Coronavirus

The nucleocapsid protein of SARS coronavirus is a protein composed of422 amino acids. It has been reported that most of the nucleocapsidproteins of other coronavirus on which much research has been conductedserve as an antigen. Such antigenic site has been intensively studied touse a target antigen of a vaccine to prevent the infection ofcoronavirus.

Therefore, sites capable of showing antigenicity in the amino acids ofthe nucleocapsid protein of SARS coronavirus was chosen by comparativeanalysis of proteins with the nucleocapsid protein of swinetransmissible gastroenteritis (TGE) coronavirus and synthesized.

Concretely, the relation between the nucleocapsid protein of swinetransmissible gastroenteritis virus and the nucleocapsid protein of SARScoronavirus was analyzed by hydrophilicity plot according to theKyte-Doolittle method, antigenic index according to the Jameson-wolfmethod and surface probability plot according to the Emini method andSARS NA and SARS NB were selected from the sequence of the nucleocapsidprotein of SARS coronavirus Tor2 isolate (FIG. 2).

Firstly, based on the sequence of the nucleocapsid protein of SARScoronavirus Tor2 isolate (28120-29388 bases, 422 amino acids), of whichthe whole sequence had been identified, the 2 to 157 amino acid sitewhich was expected to be an antigenic site was selected and denominatedSARS NA and the 163 to 305 amino acid site was selected and denominatedSARS NB. In the present invention, the gene of the SARS NB site wassynthesized.

In order to synthesize a gene corresponding to the 143 length aminoacids denominated SARS NB, PCR was performed using primers of SEQ IDNOs: 17 to 26 to obtain the amplified SARS NB gene of 429 bp. SEQ ID NO:17: 5′-ggatcccctcaaggtacaacattgccaaaaggcttctacgcagagggtagccgtgg-3′ SEQID NO: 18: 5′-accacgactacgtgatgaagaacgagaagaggcttgactgccgccacggctacc-3′SEQ ID NO: 19:5′-cacgtagtcgtggtaattcacgtaattcaactcctggcagcagtcgtggtaat-3′ SEQ ID NO:20: 5′-gcgagggcagtttcaccaccaccgctagccatacgagcaggagaattaccacga-3′ SEQ IDNO: 21: 5′-gaaactgccctcgcacttttgctgcttgaccgtttgaaccagcttgagagcaa-3′ SEQID NO: 22: 5′-tagtgacagtttgaccttgttgttgttggcctttaccagaaactttgctctcaa-3′SEQ ID NO: 23:5′-caaactgtcactaagaaatctgctgctgaggcatctaaaaagcctcgtcaaaaacgt-3′ SEQ IDNO: 24:5′-ggaccacgacgcccaaatgcttgagtgacgttgtactgttttgtggcagtacgtttttg-3′ SEQ IDNO: 25: 5′-gggcgtcgtggtccagaacaaacccaaggtaatttcggggaccaagaccttatccgt-3′SEQ ID NO: 26:5′-ggtaccaagcttattaaatttgcggccaatgtttgtaatcagtaccttgacggataagg-3′

In addition, the genes of the antigenic sites were obtained bysynthesis, a gene encoding the site of 2 to 305 amino acids wasamplified by PCR using the SARS nucleocapsid cDNA clone (SARScoronavirus TOR2) from Canada's Michael Smith Genome Science Center as atemplate and primers of SEQ ID NOs: 27 and 28 to obtain a gene of 912bp, which was denominated SARS N. SEQ ID NO: 27 (N sense):5′-cgcggatcctctgataatggtccgcaa-3′ SEQ ID NO: 28 (N anti-sense):5′-cggggtaccttaaatttgcggccaatgttt-3′

EXAMPLE 3 Construction of pHCE2LB:pgsA-SARS SA and pHCE2LB:pgsA-SARS SCVectors for Surface Expression

The surface expression vectors pHCE2LB:pgsA-SARS SA andpHCE2LB:pgsA-SARS SC capable of surface expressing the antigenic sitesSARS SA and SC in the spike protein of SARS coronavirus were constructedusing pgsA of the gene (pgsBCA) of the cellular outer membrane proteinderived from Bacillus genus strain and participating in the synthesis ofpoly-gamma-glutamic acid and a gram negative microorganism and a grampositive microorganism as hosts.

Firstly, in order to introduce the antigenic sites SARS SA and SARS SCin the spike protein of SARS coronavirus to a vector for surfaceexpression having the L1 antigen of human papilloma virus expressed withgram negative and gram positive microorganisms as hosts (a vectorcontaining HCE promoter, which is a constantly high expression promoter,pgsA of the gene (pgsBCA) of the cellular outer membrane proteinparticipating in the synthesis of poly-gamma-glutamic acid and HPV L1 inpAT which is a vector for general use for gram negative and grampositive bactera), pHCE2LB:pgsA-HPVL1 (KCTC 10349BP) was digested withBamHI and KpnI. The HPVL1 gene was removed to prepare a vectorpHCE2LB:pgsA for surface expression.

The SARS SA and SARS SC antigen genes synthesized in Example 1 were eachdigested with restriction enzymes BamHI and KpnI and joined to theC-terminal region of the gene pgsA of the cellular outer membraneprotein participating in the synthesis of poly-gamma-glutamic acid ofthe previously prepared surface expression vector pHCE2LB:pgsA inaccordance with the translation codon to prepare vectorspHCE2LB:pgsA-SARS SA and pHCE2LB:pgsA-SARS SC (FIGS. 3A and 3B). Thegram positive bacterium Lactobacillus was transformed with the preparedsurface expression vectors pHCE2LB:pgsA-SARS SA and pHCE2LB:pgsA-SARSSC, and the presence of pHCE2LB:pgsA-SARS SA and pHCE2LB:pgsA-SARS SCplasmids in Lactobacillus was examined.

EXAMPLE 4 Construction of pHCE2LB:pgsA:SARS SBC Vector for SurfaceExpression

The pHCE2LB:pgsA-SARS SBC vector capable of surface expressing theantigenic site SARS SBC in the spike protein of SARS coronavirus wasconstructed using pgsA of the gene (pgsBCA) of the cellular outermembrane protein derived from Bacillus genus strain and participating inthe synthesis of poly-gamma-glutamic acid.

Firstly, by the method described in the Example 3, the surfaceexpression vector pHCE2LB:pgsA was prepared. The gene encoding the264-596 amino acid site was amplified by PCR using the SARS spike cDNAclone of SARS coronavirus, described in the Example 1, as a template toobtain SARS SBC gene of 996 bp. The SARS SBC gene was then inserted intothe surface expression vector pHCE2LB:pgsA to prepare pHCE2LB:pgsA-SARSSBC (FIG. 3C). The gram positive bacterium Lactobacillus was transformedwith the prepared surface expression vector pHCE2LB:pgsA-SARS SBC andthe presence of pHCE2LB:pgsA-SARS SBC plasmid in Lactobacillus wasexamined.

EXAMPLE 5 Construction of pHCE2LB:pgsA:SARS NB Vector for SurfaceExpression

The pHCE2LB:pgsA-SARS NB vector capable of surface expressing theantigenic site SARS NB in the nucleocapsid protein of SARS coronaviruswas constructed using pgsA of the gene (pgsBCA) of the cellular outermembrane protein derived from Bacillus genus strain and participating inthe synthesis of poly-gamma-glutamic acid.

Firstly, by the method described in the Example 3, the surfaceexpression vector pHCE2LB:pgsA was prepared. The SARS NB antigen genesynthesized in the Example 2 was digested with restriction enzymes BamHIand KpnI and joined to the C-terminal of the gene pgsA of the cellularouter membrane protein participating in the synthesis ofpoly-gamma-glutamic acid of the previously prepared surface expressionvector pHCE2LB:pgsA in accordance with the translation codon to preparea vector pHCE2LB:pgsA-SARS NB (FIG. 4A). The gram positive bacteriumLactobacillus was transformed with the prepared surface expressionvector pHCE2LB:pgsA-SARS NB and the presence of pHCE2LB:pgsA-SARS NBplasmid in Lactobacillus was examined.

EXAMPLE 6 Construction of pHCE2LB:pgsA-SARS N Vector for SurfaceExpression

The pHCE2LB:pgsA-SARS N vector capable of surface expressing theantigenic site SARS N in the nucleocapsid protein of SARS coronaviruswas constructed using pgsA of the gene (pgsBCA) of the cellular outermembrane protein derived from Bacilius genus strain and participating inthe synthesis of poly-gamma-glutamic acid.

Firstly, by the method described in the Example 3, the surfaceexpression vector pHCE2LB:pgsA was prepared. The gene encoding the 2305amino acid site was amplified by PCR using the SARS nucleocapsid cDNAclone of SARS coronavirus, described in the Example 2, as a template toobtain SARS N gene of 912 bp. The SARS N gene was then inserted into thesurface expression vector pHCE2LB:pgsA to prepare pHCE2LB:pgsA-SARS N(FIG. 4B). The gram positive bacterium Lactobacillus was transformedwith the prepared surface expression vector pHCE2LB:pgsA-SARS N and thepresence of pHCE2LB:pgsA-SARS N plasmid in Lactobacillus was examined.

EXAMPLE 7 Confirmation of Surface Expression of SARS Virus Spike AntigenProtein on Lactic Acid Bacteria

Lactobacillus was transformed with the surface expression vectorspHCE2LB:pgsA-SARS SA, pHCE2LB:pgsA-SARS SC and pHCE2LB:pgsA-SARS SBC andexamined for expression of respective antigen proteins.

The expression of the antigenic sites in the spike antigen of SARS virusfused with the C-terminal of the gene pgsA synthesizingpoly-gamma-glutamic acid was induced by transforming Lactobacillus caseiwith pHCE2LB:pgsA-SARS SA, pHCE2LB:pgsA-SARS SC and pHCE2LB:pgsA-SARSSBC, subjecting the transformed strain in MRS medium (Lactobacillus MRS,Becton Dickinson and Company Sparks, USA), to a stationary culture andmultiplication at 37° C.

The expression of each spike antigen was identified by performingWestern immunoblotting using SDS-polyacrylamide gel electrophoresis anda specific antibody to pgsA. The whole cells of Lactobacillus caseiwhose expression is induced concretely were denatured with proteinsobtained at the same cell concentration to prepare samples. They wereanalyzed by SDS-polyacrylamide gel electrophoresis and the fractionatedproteins were transferred to PVDF membrane (polyvinylidene-difluoridemembranes, Bio-Rad). The PVDF membrane with the proteins transferredthereon in a blocking buffer solution (50 mM Tris HCl, 5% skim milk, pH8.0) was blocked by shaking for 1 hour and reacted with rabbit-derivedpolyclone primary antibody to pgsA, which have been diluted 1000 timeswith the blocking buffer solution, for 12 hours.

After completion of the reaction, the membrane was washed with buffersolution and reacted with biotin-binding secondary antibody to rabbit,which have been diluted 1000 times with the blocking buffer solution,for 4 hours. After completion of the reaction, the membrane was washedwith buffer solution and reacted with a avidin-biotin reagent for 1hour, followed by washing. The washed membrane was treated with H₂O₂ andDAB solution as a substrate and a color developing agent to confirm thatthe specific bonding between the specific antibody to pgsA and thefusion protein (FIG. 5). In FIG. 5A, lane 1 is non-transformedLactobacillus casei, and lane 2, 3 and 4 are Lactobacillus caseitransformed with pHCE2LB:pgsA-SARS SA. In FIG. 5B, lane 1 isnon-transformed Lactobacillus casei, and lane 2, 3, 4, 5 and 6 areLactobacillus casei transformed with pHCE2LB:pgsA-SARS SC/. In FIG. 5C,lane 1 is non-transformed Lactobacillus casei, and lane 2 isLactobacillus casei transformed with pHCE2LB:pgsA-SARS SBC.

As shown in FIG. 5, specific fusion proteins [pgsA-SARS SA of about54kDa (FIG. 5A), pgsA-SARS SC of about 51kDa (FIG. 5B) and pgsA-SARS SBCof about 78 kDa (FIG. 5C)] were identified in the whole cell ofrespective lactic acid bacteria.

Also, in order to confirm if respective antigen proteins were expressedwith pgsA in the lactic acid bacteria transformed by thepHCE2LB:pgsA-SARS SA and pHCE2LB:pgsA-SARS:SBC surface expressionvectors on the surface, the lactic acid bacteria transformed by therespective vectors were fractionated by the cell fractionation methodusing a ultracentrifuge into the cell wall and the cytoplasm and thepositions of the respective fusion proteins were identified by Westernblot using the specific antibody to pgsA.

Concretely, Lactobacillus which had the surface expression of the fusionproteins induced by the above described method were harvested to be thesame cell concentration as non-transformed Lactobacillus. The cells werewashed several times with TES buffer (10 nM Tris-HCl, pH 8.0, 1 mM EDTA,25% sucrose), suspended in distilled water containing 5 mg/ml lysozyme,1 mM PMSF and 1 mM EDTA, frozen at −60° C. and thawed at roomtemperature several times, treated with, DNase (0.5 mg/ml) and RNase(0.5 mg/ml) and subjected to sonication for cell destruction. Then, thecell lysate was centrifuged at 4° C., for 20 minutes at 10,000× g toseparate the non-lysed whole Lactobacillus (pellet; whole cell fraction)and cellular debris (supernatant). The separated cellular debris wascentrifuged at 4° C. for 1 hour at 21,000× g to obtain the supernatant(soluble fraction) containing cytoplasm proteins of Lactobacillus andpellets. The obtained pellets were suspended in TE solution (10 mMTris-HCl, pH 8.0, 1 mM EDTA, pH 7.4) containing 1% SDS to obtain cellwall proteins (cell wall fraction) of Lactobacillus.

The respective fractions were subjected to Western immunoblotting usingSDS-polyacrylamide gel electrophoresis and the antibody to pgsA antigento confirm that the spike antigens of SARS virus fused with pgsA existedin the cell wall, among the respective Lactobacillus fractions (FIG. 6).In FIG. 6A, lane 1 is non-transformed Lactobacillus casei, lane 2 is thewhole cells of Lactobacillus casei transformed with pHCE2LB:pgsA-SARSSA, lane 3 and 4 are the soluble fraction and the cell wall fraction ofthe strain trasformed with pHCE2LB:pgsA-SARS SA, respectively. In FIG.6B, lane 1 is non-transformed Lactobacillus casei, lane 2 is the wholecells of Lactobacillus casei transformed with pHCE2LB:pgsA-SARS SBC,lane 3 and 4 are the soluble fraction and the cell wall fraction of thestrain trasformed with. pHCE2LB:pgsA-SARS SBC, respectively.

As shown in FIG. 6, the SARS SA protein of about 54 kDa fused with pgsAand the SARS SBC protein of about 78 kDa fused with pgsA were identifiedin the whole cell and the cell wall fraction of lactic acid bacteria.From these results, it was noted that the respective SARS antigenproteins fused with pgsA were expressed and placed by migrating to thesurface of lactic acid bacteria by pgsA.

Also, by fluorescence-activating cell sorting (FACS) flow cytometry, itwas identified that the expression of the antigen group of the spikeantigen of SARS virus took place on the surface of Lactobacillus by thefusion with C-terminal of the poly-gamma-glutamic acid synthesizingprotein pgsA.

For immunofluorescence dying, expression induced Lactobacillus washarvested to be the same cell concentration. The cells were washedseveral times with buffer solution (PBS buffer, pH 7.4), suspended in 1ml of buffer solution containing 1% bovine serum albumin and reactedwith mouse-derived polyclone primary antibody to the spike antigen ofSARS virus, which have been diluted 1000 times, at 4° C. for 12 hours.After completion of the reaction, the cells were washed several timeswith buffer solution, suspended in 1 ml of buffer solution containing 1%bovine serum albumin and reacted with biotin-binding secondary antibody,which have been diluted 1000 times, at 4° C. for 3 hours. Again, aftercompletion of the reaction, the cells were washed several times withbuffer solution, suspended in 0.1 ml of buffer solution containing 1%bovine serum albumin and bound to streptavidin-R-phycoerythrin dye agentspecific to biotin, which have been diluted 1000 times.

After completion of the reaction, Lactobacillus was washed severaltimes, and examined by fluorescence-activating cell sorting (FACS) flowcytometry. It was noted that as compared to non-transformedLactobacillus, the SBC spike antigen protein of SARS virus was expressedon the surface of Lactobacillus (FIG. 6C). In FIG. 6C, the grey part isderived from non-transformed Lactobacillus casei and the white part isderived from transformed pHCE2LB:pgsA-SARS SBC/Lactobacillus casei. Asshown in FIG. 6C, it was clearly noted that the SBC spike antigenprotein was surface expressed in lactic acid bacteria transformed withpHCE2LB:pgsA-SARS SBC vector while no fluorescence expression wasobserved in non-transformed Lactobacillus casei.

EXAMPLE 8 Confirmation of Surface Expression of SARS Virus NucleocapsidAntigen Protein on Lactic Acid Bacteria

Lactobacillus was transformed with the surface expression vectorspHCE2LB:pgsA-SARS NB and pHCE2LB:pgsA-SARS N and examined for expressionof respective antigen proteins.

The expression of the antigenic sites in the nucleocapsid antigen ofSARS virus fused respectively with the C-terminal of the gene pgsAsynthesizing poly-gamma-glutamic acid was induced by transformingLactobacillus casei with pHCE2LB:pgsA-SARS NB and pHCE2LB:pgsA-SARS Nrespectively, subjecting the transformed strain in MRS medium(Lactobacillus MRS, Becton Dickinson and Company Sparks, USA), to astationary culture and multiplication at 37° C.

In order to confirm if respective antigen proteins were expressed withpgsA in the lactic acid bacteria transformed by the pHCE2LB:pgsA-SARS NBand pHCE2LB:pgsA-SARS N surface expression vectors on its surface, thelactic acid bacteria transformed with each vector by the same method asin the Example 7 were fractionated by the cell fractionation methodusing a ultracentrifuge into the cell wall and the cytoplasm and thepositions of the respective fusion proteins were identified by Westernblot using the specific antibody to pgsA.

As a result, The respective fractions were subjected to Westernimmunoblotting using SDS-polyacrylamide gel electrophoresis and theantibody to pgsA antigen to confirm that the nucleo antigens of SARSvirus fused with pgsA existed in the cell wall, among the respectiveLactobacillus fractions (FIG. 7). In FIG. 7A, lane 1 is non-transformedLactobacilius casei, lane 2 is the whole cell of transformedpHCE2LB:pgsA-SARS NB/Lactobacillus casei, lane 3 and 4 are the solublefraction and the cell wall fraction of the strain trasformed withpHCE2LB:pgsA-SARS NB, respectively. In FIG. 7B, lane 1 isnon-transformed Lactobacillus casei, lane 2 is the whole cell of thetransformed pHCE2LB:pgsA-SARS N/Lactobacillus casei, lane 3 and 4 arethe soluble fraction and the cell wall fraction of the strain trasformedwith pHCE2LB:pgsA-SARS N, respectively.

As shown in FIG. 7, the SARS NB protein of about 57 kDa fused with pgsAand the SARS N protein of about 75 kDa fused with pgsA were identifiedin the whole cell and the cell wall fraction of lactic acid bacteria.From these results, it was noted that the respective SARS antigenproteins fused with pgsA were expressed and placed by migrating to thesurface of lactic acid bacteria by pgsA.

EXAMPLE 9 Analysis of Vaccine Effect of Lactic Acid Bacteria with SpikeAntigen Protein and Nucleocapsid Antigen Protein of SARS Virus SurfaceExpressed

Gram positive bacterium Lactobacillus casei was transformed with thesurface expression vectors pHCE2LB:pgsA-SARS SA, pHCE2LB:pgsA-SARS SCand pHCE2LB:pgsA-SARS NB, prepared in the foregoing Examples andexpression of the antigens on the surface of Lactobacillus casei wasinduced. The antigenicity of the spike antigen protein and nucleocapsidantigen protein of SARS virus fuged with cellular outer membrane proteinpgsA participating poly-gamma-glutamic acid synthesis was examined usinga mouse model.

Concretely, Lactobacillus casei was transformed with the surfaceexpression vectors pHCE2LB:pgsA-SARS SA, pHCE2LB:pgsA-SARS SC andpHCE2LB:pgsA-SARS NB according to the present invention. The cells wereharvested to be the same cell concentration and washed several timeswith buffer solution (PBS buffer, pH 7.4). 5×10⁹ Lactobacillus cellswith the antigen surface expressed were orally administered to a 4-6week old BALB/c mouse 3 times a day every other day, 3 times a day everyother day after 1 week, 3 times a day every other day after 2 weeks, and3 times a day every other day after 4 weeks. Also, 1×10⁹ Lactobacilluscells with the antigen surface expressed were intranasally administeredto a mouse 3 times a day every other day, 3 times a day every other dayafter 1 week, 2 times a day every two days after 2 weeks, and 2 times aday every two days after 4 weeks. After oral and intranasaladmistrations, every two weeks, {circle around (1)} serum of each mousewas taken and examined for IgG antibody value to the spike antigenprotein and the nucleocapsid antigen protein in the serum and {circlearound (2)} the suspension which comes after washing the inside of theintestines from each mouse and suspension which comes after washing theinside of bronchus and alveola from each mouse were examined for IgAantibody value to the spike antigen protein and nucleocapsid antigenprotein, by ELISA.

10 BALB/c mice(4-6 week old) were assigned to one group. A mixture oflactic acid bacteria, each expressing SARS SA and SARS SC, was assignedto one group, lactic acid bacteria expressing SARS NB was assigned toone group, and a mixture of lactic acid bacteria, each expressing SARSSA, SARS SC and SARS NB, was assigned to one group. These three groupswere divided into a oral administraion group and an intranasaladministration group to make 8 groups including control group.

FIG. 8 shows the IgG antibody value to the SARS SA and SARS SC antigens,which are the spike antigen proteins of SARS virus, in serum of mice.FIG. 9 shows the IgA antibody value to the SARS SA and SARS SC antigens,which are the spike antigen proteins, in the suspension which comesafter washing the inside of the intestines and suspension which comesafter washing the inside of bronchus and alveola of mice according toELISA, in which A is the IgA antibody value of the oral administrationgroup and B is the IgA antibody value of the intranasal administrationgroup.

Also, FIG. 10 shows the IgG antibody value to the SARS NB antigen, whichis the nucleocapsid antigen protein of SARS virus, in serum of mice.FIG. 11 shows the IgA antibody value to the SARS NB antigen, which isthe nucleocapsid antigen protein of SARS virus, in the suspension whichcomes after washing the inside of the intestines and suspension whichcomes after washing the inside of bronchus and alveola of mice accordingto ELISA, in which A is the IgA antibody value of the oraladministration group and B is the IgA antibody value of the intranasaladministration group.

As shown in FIGS. 8 to 11, it was noted that the IgG antibody value andthe IgA antibody value to the antigen groups of the spike andnucleocapsid antigen proteins of SARS virus were considerably higher inin the serum, the intestine washing liquid and bronchus-alveola washingliquid of BALB/c mice administered with transformed Lactobacillus bypHCE2LB:pgsA-SARS SA, pHCE2LB:pgsA-SARS SC and pHCE2LB:pgsA-SARS NB,alone or in combination as compared to the control group.

Therefore, it was noted that the microorganism having the antigen groupsof the spike and nucleocapsid antigen proteins of SARS virus surfaceexpressed according to the present invention can be effectively used asa live vaccine.

While the present invention has been described with reference to theparticular illustrative embodiments, it is not to be restricted by theembodiments but only by the appended claims. It is to be appreciatedthat those skilled in the art can change or modify the embodimentswithout departing from the scope and spirit of the present invention.

INDUSTRIAL APPLICABILITY

As described above, the transformed microorganism expressing an antigenprotein of SARS inducing coronavirus on their surface according to thepresent invention and the antigen protein extracted and purified fromthe microorganism can be used as a vaccine for prevention and treatmentof SARS. Particularly, it is advantageously possible to economicallyproduce a vaccine for oral use using the recombinant strain expressingan SARS coronavirus antigen according to the present invention.

1. A surface expression vector comprising any one or two or more ofpgsB, pgsC and pgsA genes encoding poly-gamma-glutamic acid synthasecomplex and a gene encoding a spike antigen protein or a nucleocapsidantigen protein of SARS coronavirus.
 2. The surface expression vectoraccording to claim 1, wherein the spike antigen protein is SARS SA, SARSSB, SARS SC, SARS SD or SARS SBC.
 3. The surface expression vectoraccording to claim 1, wherein the nucleocapsid antigen protein is SARSNA, SARS NB or SARS N.
 4. The surface expression vector according toclaim 2, wherein the vector is pHCE2LB:pgsA-SARS SA, pHCE2LB:pgsA-SARSSC or pHCE2LB:pgsA-SARS SBC.
 5. The surface expression vector accordingto claim 3, wherein the vector is pHCE2LB:pgsA-SARS NB orpHCE2LB:pgsA-SARS N.
 6. A microorganism transformed by the expressionvector of claim
 1. 7. The microorganism according to claim 6 wherein themicroorganism is selected from the group consisting of E. coli,Salmonella typhi, Salmonella typhimurium, Vibrio cholerae, Mycobacteriumbovis, Shigella, Bacillus, lactic acid bacterium, Staphylococcus,Listeria monocytogenes, and Streptococcus.
 8. A method for producing aspike antigen protein or a nucleocapsid antigen protein of SARScoronavirus comprising culturing the microorganism of claim
 6. 9. Avaccine for prevention of SARS virus comprising the spike antigenprotein or the nucleocapsid antigen protein or the produced by themethod of claim 8, as an effective ingredient.
 10. The vaccine accordingto claim 9, wherein the antigen protein is an expressed form on thesurface of microorganism, a crudely extracted form or a purified form.11. The vaccine according to claim 9, wherein the vaccine is adapted tobe taken oral administration or in food.
 12. The vaccine according toclaim 9, wherein the vaccine is adapted for subcutaneous orintra-peritoneal injection.
 13. The vaccine according to claim 9,wherein the vaccine is adapted for intranasal administration.
 14. Themethod according to claim 8, wherein the microorganism is lactic acidbacterium.
 15. A lactic acid bacterium, which is produced by the methodof claim 14 having the spike antigen protein or the nucleocapsid antigenprotein of SARS coronavirus expressed on its surface.
 16. A vaccine forprevention of SARS comprising the lactic acid bacterium of claim 15, anantigen protein extracted from said lactic acid bacterium, as aneffective ingredient.
 17. The vaccine according to claim 16, wherein thevaccine is adapted to be taken by oral administration or in food. 18.The vaccine according to claim 16, wherein the vaccine is adapted forsubcutaneous or intra-peritoneal injection.
 19. The vaccine according toclaim 16, wherein the vaccine is adapted for intranasal administration.